The proposed relationship of nerve growth factor (NGF) and insulin has been extended through structural and mechanistic studies. The distribution of tryptophan in NGF in the three-dimensional structure has been determined. Trp 21 is located in the surface, fully available to solvent, Trp 99 is partially unavailable to reaction and Trp 76 is complete unreactive, presumably because it's buried in the interior of the molecule. Comparison of the positions of the corresponding residues in the three-dimensional structure of insulin reveals an entirely compatible arrangement. Furthermore, none of these residues appear to be required for formation of an NGF-receptor complex. Similar experiments with tyrosine indicate both such residues in NGF are partially buried and may exist in a hydrogen-bonded configuration such as found for the A-19 tyrosine of insulin. The presence of NGF receptors in the cell membranes of NGF-responsive neurons has been rigorously demonstrated by the use of isolubilized NGF. In order to prepare derivatives that did not release detectable amounts of soluble activity, it was necessary to couple NGF to Sepharose activated with CNBr in the presence of 6 M quanidine-HCl. Insolubilized NGF, prepared in this manner, is active biologically demonstrating that there is no requirement for the protein to be taken up by responsive neurons to express biological activity and clearly indicates the presence of cell surface receptors. The binding of I125-NGF to these receptors has been measured with a variety of tissues. All responsive tissues examined show specific binding but to varying degrees. Organs receiving sympathetic innervation also show low but significant binding. The NGF from Naja naja (cobra) venom has been isolated and purified to homogeneity. It possesses a dimeric structure essentially identical to mouse NGF.